Fig. 7.

shRNA-mediated Myo1c knockdown inhibits Neph1 membrane localization. (A) Myo1c knockdown was induced by the transfection of a plasmid encoding Myo1c shRNA in cultured human podocytes. Stable transfectants were selected, and the extent of the Myo1c protein knockdown was assessed by Western blotting. (B) Control and knockdown cells were analyzed by immunofluorescence using Myo1c (Alexa 568) and Neph1 (Alexa 488) antibodies and phalloidin (Alexa 488). (C) Quantitation of Myo1c knockdown from five different experiments is represented as the mean pixel intensity of Myo1c. (D and E) Mean pixel intensity analysis of Myo1c and Neph1 at the podocyte cell membrane from five different experiments (15 cells each) was performed and shows a significant reduction in the amount of Neph1 at the cell membrane compared to control shRNA (P < 0.001). (F) Surface Neph1 was labeled in Myo1c knockdown and control cells with Neph1 extracellular antibody under unpermeabilized conditions. Immunofluorescence analysis was performed by using confocal imaging. (G) Quantitative analysis of single-plane images (n = 10 cells) shows a significant decrease in the mean pixel intensity of surface Neph1 labeling in Myo1c knockdown cells compared to control cells. (H) Immunofluorescence analysis of Myo1c knockdown cells with ZO-1 antibody shows that ZO-1 localization remains unchanged compared to control shRNA. (I) Lysates from control and Myo1c knockdown cells were Western blotted with Myo1c, Neph1, and Myo1e antibodies and show that Myo1c knockdown does not alter Neph1 and Myo1e protein levels. (J) Myo1c and control knockdown cells were fractionated to isolate the membrane fractions, which were analyzed by Western blotting using Neph1 antibody.